VL30 retrotransposition is associated with induced EMT, CSC generation and tumorigenesis in HC11 mouse mammary stem‑like epithelial cells

Abstract

Retrotransposons copy their sequences via an RNA intermediate, followed by reverse transcription into cDNA and random insertion, into a new genomic locus. New retrotransposon copies may lead to cell transformation and/or tumorigenesis through insertional mutagenesis. Methylation is a major defense mechanism against retrotransposon RNA expression and retrotransposition in differentiated cells, whereas stem cells are relatively hypo-methylated. Epithelial-to-mesenchymal transition (EMT), which transforms normal epithelial cells into mesenchymal-like cells, also contributes to tumor progression and tumor metastasis. Cancer stem cells (CSCs), a fraction of undifferentiated tumor-initiating cancer cells, are reciprocally related to EMT. In the present study, the outcome of long terminal repeat (LTR)-Viral-Like 30 (VL30) retrotransposition was examined in mouse mammary stem-like/progenitor HC11 epithelial cells. The transfection of HC11 cells with a VL30 retrotransposon, engineered with an EGFP-based retrotransposition cassette, elicited a higher retrotransposition frequency in comparison to differentiated J3B1A and C127 mouse mammary cells. Fluorescence microscopy and PCR analysis confirmed the specificity of retrotransposition events. The differentiated retrotransposition-positive cells retained their epithelial morphology, while the respective HC11 cells acquired mesenchymal features associated with the loss of E-cadherin, the induction of N-cadherin, and fibronectin and vimentin protein expression, as well as an increased transforming growth factor (TGF)-β1, Slug, Snail-1 and Twist mRNA expression. In addition, they were characterized by cell proliferation in low serum, and the acquisition of CSC-like properties indicated by mammosphere formation under anchorage-independent conditions. Mammospheres exhibited an increased Nanog and Oct4 mRNA expression and a CD44 + /CD24 -/low antigenic phenotype, as well as self-renewal and differentiation capacity, forming mammary acini-like structures. DNA sequencing analysis of retrotransposition-positive HC11 cells revealed retrotransposed VL30 copies integrated at the vicinity of EMT-, cancer type-and breast cancer-related genes. The inoculation of these cells into Balb/c mice produced cytokeratin-positive tumors containing pancytokeratin-positive cells, indicative of cell invasion features. On the whole, the findings of the present study demonstrate, for the first time, to the best of our knowledge, that stem-like epithelial HC11 cells are amenable to VL30 retrotransposition associated with the induction of EMT and CSC generation, leading to tumorigenesis.

Tinzaparin inhibits VL30 retrotransposition induced by oxidative stress and/or VEGF in HC11 mouse progenitor mammary cells: Association between inhibition of cancer stem cell proliferation and mammosphere disaggregation

“…Furthermore, microscopic observation revealed two common VL30 retrotransposition-derived features, as shown for representative cl.11 ( Fig. S3 ): i) An expected VL30 retrotransposition-induced EMT phenotype ( 46 ) observed at low confluence ( Fig. S3B ); and ii) cytoplasmic EGFP fluorescence ( Fig.…”

“…In a previous study, VL30 retrotransposition-positive HC11 cell clones were generated, exhibiting an induced EMT phenotype, CSC properties and solid tumor production in syngeneic Balb/c mice ( 46 ), and with a retrotransposition frequency of ~2-5.5%. Notably, as HC11 cells possess stem-like properties, the occurrence of retrotransposition events could be explained by their hypomethylation status ( 46 ), as has been suggested for human stem cells ( 53 ).…”

“…In a previous study, VL30 retrotransposition-positive HC11 cell clones were generated, exhibiting an induced EMT phenotype, CSC properties and solid tumor production in syngeneic Balb/c mice ( 46 ), and with a retrotransposition frequency of ~2-5.5%. Notably, as HC11 cells possess stem-like properties, the occurrence of retrotransposition events could be explained by their hypomethylation status ( 46 ), as has been suggested for human stem cells ( 53 ). To examine the effect of tinzaparin and/or VEGF treatment on VL30 retrotransposition, new clones were isolated with a retrotransposition frequency of 7–15%, allowing a more accurate measurement of retrotransposition changes, especially in the case of inhibition.…”

“…Using both the previously well-characterized HC11 cl.19 for its retrotransposition-induced CSCs and mammosphere formation properties ( 46 ), and HC11 cl.15 from the present study, the effect of tinzaparin on HC11 cell proliferation rate was initially examined. Trypsinized cells from these clones were subjected to real-time cell analysis in the absence or presence of 2 IU/ml tinzaparin for up to 80 h, using normal HC11 cells as the control.…”

“…Previously, VL30 retrotransposition in mouse HC11 epithelial mammary cells (with stem-like properties) was reported in association with induced EMT, CSC generation and tumor growth ( 46 ). Since tinzaparin modulates the expression of a large number of genes ( 17 ), and LTR-retrotransposition is accomplished through a retrotransposon RNA-intermediate, the aim of the present study was to establish whether tinzaparin also affected VL30 retrotransposition.…”